The objective of the 5 year career development plan outlined in this KO1 application is to foster the development of the candidate, Dr. Alford into a productive, independent investigator in the field of osteoblast cellular and extracellular matrix biology. Under the guidance of her mentors, Drs Steven A Goldstein and Renny T Franceschi, and through regular interactions with expert consultants, Stephen J. Weiss and Kurt D. Hankenson, the candidate will carry out the proposed research as a member of the Orthopaedic Research Laboratories in the Department of Orthopaedic Surgery at the University of Michigan. In the 5 year research plan proposed here, we will utilize primary MSC harvested from TSP2-null mice and their littermates in a well established in vitro model of osteoblastogenesis to test the global hypothesis that matrix-bound TSP2 makes specific contributions to osteoblast lineage progression which are dependent on the differentiation state of the cell, as well as on the unique extracellular milieu associated with matrix assembly, maturation and mineralization. Specifically, in aim 1 we will address the working hypothesis that in the context of early osteoblast-differentiation events, TSP2 promotes the assembly of a type I collagen-rich osteoblast-derived extracellular matrix, and as a result, it indirectly promotes collagen-dependent signal transduction events that are critical for osteoblast differentiation. In specific aim 2, we will address the premise that, in the context of matrix mineralization, TSP2 makes an additional unique contribution to osteoblast maturation. Specifically, we hypothesize that TSP2 is proteolytically processed to release biologically relevant fragments that affect matrix mineralization. Together, the experiments outlined in this proposal will define differentiation-stage dependent contributions that TSP2 makes to osteoblast lineage progression and matrix mineralization. PUBLIC HEALTH RELEVANCE: A full understanding of how biochemical and physiological context influence the function of ECM molecules, such as thrombospondin-2, in marrow stromal cell lineage progression and osteoblast differentiation are essential in the design of therapeutics aimed at manipulating the environment of osteoblast precursors in order to promote bone formation under conditions of skeletal injury, aging or disease.